Dataset Information

Heterogeneous calretinin expression in the avian cochlear nucleus angularis.

ABSTRACT: Multiple calcium-binding proteins (CaBPs) are expressed at high levels and in complementary patterns in the auditory pathways of birds, mammals, and other vertebrates, but whether specific members of the CaBP family can be used to identify neuronal subpopulations is unclear. We used double immunofluorescence labeling of calretinin (CR) in combination with neuronal markers to investigate the distribution of CR-expressing neurons in brainstem sections of the cochlear nucleus in the chicken (Gallus gallus domesticus). While CR was homogeneously expressed in cochlear nucleus magnocellularis, CR expression was highly heterogeneous in cochlear nucleus angularis (NA), a nucleus with diverse cell types analogous in function to neurons in the mammalian ventral cochlear nucleus. To quantify the distribution of CR in the total NA cell population, we used antibodies against neuronal nuclear protein (NeuN), a postmitotic neuron-specific nuclear marker. In NA neurons, NeuN label was variably localized to the cell nucleus and the cytoplasm, and the intensity of NeuN immunoreactivity was inversely correlated with the intensity of CR immunoreactivity. The percentage of CR?+?neurons in NA increased from 31 % in embryonic (E)17/18 chicks, to 44 % around hatching (E21), to 51 % in postnatal day (P) 8 chicks. By P8, the distribution of CR?+?neurons was uniform, both rostrocaudal and in the tonotopic (dorsoventral) axis. Immunoreactivity for the voltage-gated potassium ion channel Kv1.1, used as a marker for physiological type, showed broad and heterogeneous postsynaptic expression in NA, but did not correlate with CR expression. These results suggest that CR may define a subpopulation of neurons within nucleus angularis.

SUBMITTER: Bloom S

PROVIDER: S-EPMC4141438 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Heterogeneous calretinin expression in the avian cochlear nucleus angularis.

Bloom S S Williams A A MacLeod K M KM

Journal of the Association for Research in Otolaryngology : JARO 20140422 4

Multiple calcium-binding proteins (CaBPs) are expressed at high levels and in complementary patterns in the auditory pathways of birds, mammals, and other vertebrates, but whether specific members of the CaBP family can be used to identify neuronal subpopulations is unclear. We used double immunofluorescence labeling of calretinin (CR) in combination with neuronal markers to investigate the distribution of CR-expressing neurons in brainstem sections of the cochlear nucleus in the chicken (Gallus ...[more]

PMID: 24752525

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Project description:Calretinin (CALB2) is a diagnostic and prognostic marker in malignant pleural mesothelioma (MPM). We previously reported that calretinin expression is regulated at the mRNA level. The presence of a medium-sized (573 nucleotide) 3' untranslated region (3'UTR) predicted to contain binding sites for miR-30a/b/c/d/e and miR-9 as well as an adenine/uridine-rich element (ARE) in all three transcripts arising from the CALB2 gene, suggests that calretinin expression is regulated via posttranscriptional mechanisms. Our aim was to investigate the role of the CALB2-3'UTR in the posttranscriptional regulation of calretinin expression in MPM. CALB2-3'UTR was inserted downstream of the luciferase reporter gene using pmiRGLO vector and reporter expression was determined after transfection into MPM cells. Targeted mutagenesis was used to generate variants harboring mutated miR-30 family and ARE binding sites. Electrophoretic mobility shift assay was used to test for the presence of ARE binding proteins. CALB2-3'UTR significantly decreased luciferase activity in MPM cells. Analysis of mutation in the ARE site revealed a further destabilization of the reporter and human antigen R (HuR) binding to the ARE sequence was detected. The mutation of two miR-30 binding sites abolished CALB2-3'UTR destabilization effect; a transient delivery of miR-30e-5p mimics or anti-miR into MPM cells resulted in a significant decrease/increase of the luciferase reporter expression and calretinin protein, respectively. Moreover, overexpression of CALB2-3'UTR quenched the effect of miR-30e-5p mimics on calretinin protein levels, possibly by sequestering the mimics, thereby suggesting a competitive endogenous RNA network. Finally, by data mining we observed that expression of miR-30e-5p was negatively correlated with the calretinin expression in a cohort of MPM patient samples. Our data show the role of (1) adenine-uridine (AU)-binding proteins in calretinin stabilization and (2) miR-30e-5p in the posttranscriptional negative regulation of calretinin expression via interaction with its 3'UTR. Furthermore, our study demonstrates a possible physiological role of calretinin's alternatively spliced transcripts.

Project description:Schizophrenia (SCH) and autism spectrum disorder (ASD) share several common aetiological and symptomatic features suggesting they may be included in a common spectrum. For example, recent results suggest that excitatory/inhibitory imbalance is relevant in the etiology of SCH and ASD. Numerous studies have investigated this imbalance in regions like the ventromedial and dorsolateral prefrontal cortex (DLPFC). However, relatively little is known about neuroanatomical changes that could reduce inhibition in subcortical structures, such as the caudate nucleus (CN), in neuropsychiatric disorders. We recently showed a significant decrease in calretinin-immunopositive (CR-ip) interneuronal density in the CN of patients with ASD without significant change in the density of neuropeptide Y-immunopositive (NPY-ip) neurons. These subtypes together constitute more than 50% of caudate interneurons and are likely necessary for maintaining excitatory/inhibitory balance. Consequently, and since SCH and ASD share characteristic features, here we tested the hypothesis, that the density of CR-ip neurons in the CN is decreased in patients with SCH. We used immunohistochemistry and qPCR for CR and NPY in six patients with schizophrenia and six control subjects. As expected, small, medium and large CR-ip interneurons were detected in the CN. We found a 38% decrease in the density of all CR-ip interneurons (P < 0.01) that was driven by the loss of the small CR-ip interneurons (P < 0.01) in patients with SCH. The densities of the large CR-ip and of the NPY-ip interneurons were not significantly altered. The lower density detected could have been due to inflammation-induced degeneration. However, the state of microglial activation assessed by quantification of ionized calcium-binding adapter molecule 1 (Iba1)- and transmembrane protein 119 (TMEM119)-immunopositive cells showed no significant difference between patients with SCH and controls. Our results warrant further studies focussing on the role of CR-ip neurons and on the striatum being a possible hub for information selection and regulation of associative cortical fields whose function have been altered in SCH.

Dataset Information

Heterogeneous calretinin expression in the avian cochlear nucleus angularis.

Publications

Heterogeneous calretinin expression in the avian cochlear nucleus angularis.

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